Finding Expandable Induced Cardiovascular Progenitor Cells
2016; 119 (1): 16-20
Induced pluripotent stem cells: at the heart of cardiovascular precision medicine
NATURE REVIEWS CARDIOLOGY
2016; 13 (6): 333-349
Reprogramming and transdifferentiation for cardiovascular development and regenerative medicine: where do we stand?
EMBO MOLECULAR MEDICINE
2015; 7 (9): 1090-1103
The advent of human induced pluripotent stem cell (hiPSC) technology has revitalized the efforts in the past decade to realize more fully the potential of human embryonic stem cells for scientific research. Adding to the possibility of generating an unlimited amount of any cell type of interest, hiPSC technology now enables the derivation of cells with patient-specific phenotypes. Given the introduction and implementation of the large-scale Precision Medicine Initiative, hiPSC technology will undoubtedly have a vital role in the advancement of cardiovascular research and medicine. In this Review, we summarize the progress that has been made in the field of hiPSC technology, with particular emphasis on cardiovascular disease modelling and drug development. The growing roles of hiPSC technology in the practice of precision medicine will also be discussed.
View details for DOI 10.1038/nrcardio.2016.36
View details for Web of Science ID 000376196300005
View details for PubMedID 27009425
A Titratable Two-Step Transcriptional Amplification Strategy for Targeted Gene Therapy Based on Ligand-Induced Intramolecular Folding of a Mutant Human Estrogen Receptor
MOLECULAR IMAGING AND BIOLOGY
2014; 16 (2): 224-234
Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options.
View details for DOI 10.15252/emmm.201504395
View details for Web of Science ID 000360963900002
View details for PubMedID 26183451
Noninvasive Imaging of Hypoxia-Inducible Factor-1 alpha Gene Therapy for Myocardial Ischemia
HUMAN GENE THERAPY METHODS
2013; 24 (5): 279-288
Molecular imaging: The key to advancing cardiac stem cell therapy
TRENDS IN CARDIOVASCULAR MEDICINE
2013; 23 (6): 201-210
The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ERG521T) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.
View details for DOI 10.1007/s11307-013-0673-4
View details for Web of Science ID 000332828200010
View details for PubMedID 23955099
Noninvasive Monitoring of Oxidative Stress in Transplanted Mesenchymal Stromal Cells
2013; 6 (7): 795-802
Cardiac stem cell therapy continues to hold promise for the treatment of ischemic heart disease despite the fact that early promising pre-clinical findings have yet to be translated into consistent clinical success. The latest human studies have collectively identified a pressing need to better understand stem cell behavior in humans and called for more incorporation of noninvasive imaging techniques into the design and evaluation of human stem cell therapy trials. This review discusses the various molecular imaging techniques validated to date for studying stem cells in living subjects, with a particular emphasis on their utilities in assessing the acute retention and the long-term survival of transplanted stem cells. These imaging techniques will be essential for advancing cardiac stem cell therapy by providing the means to both guide ongoing optimization and predict treatment response in humans.
View details for DOI 10.1016/j.tcm.2012.12.003
View details for Web of Science ID 000322147600003
View details for PubMedID 23561794
Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer
MOLECULAR IMAGING AND BIOLOGY
2011; 13 (6): 1124-1132
OBJECTIVES: The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells. BACKGROUND: In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function. METHODS: Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions. RESULTS: After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor. CONCLUSIONS: Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.
View details for DOI 10.1016/j.jcmg.2012.11.018
View details for Web of Science ID 000321677300006
View details for PubMedID 23643284
Cardiovascular Molecular Imaging Focus on Clinical Translation
2011; 123 (4): 425-443
Longitudinal, Noninvasive Imaging of T-Cell Effector Function and Proliferation in Living Subjects
2010; 70 (24): 10141-10149
We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1? (HIF-1?-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.The plasmid pUbi-hrl-pUbi-HIF-1?-VP2, which expresses both hrl and HIF-1?-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1?-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).Strong correlations existed between the expression of hRL and each of HIF-1?-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1?-VP2 transgene expression were successfully imaged using BLI.An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.
View details for DOI 10.1007/s11307-011-0471-9
View details for Web of Science ID 000296794400009
View details for PubMedID 21267661
Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy
2010; 17 (7): 827-838
Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.
View details for DOI 10.1158/0008-5472.CAN-10-1843
View details for Web of Science ID 000285334200016
View details for PubMedID 21159636
Antioxidants Improve Early Survival of Cardiomyoblasts After Transplantation to the Myocardium
MOLECULAR IMAGING AND BIOLOGY
2010; 12 (3): 325-334
Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.
View details for DOI 10.1038/gt.2010.30
View details for Web of Science ID 000279614600002
View details for PubMedID 20237511
Visualizing Implanted Tumors in Mice with Magnetic Resonance Imaging Using Magnetotactic Bacteria
CLINICAL CANCER RESEARCH
2009; 15 (16): 5170-5177
We tested the hypothesis that modulation of the microenvironment (using antioxidants) will increase stem cell survival in hypoxia and after transplantation to the myocardium.Rat cardiomyoblasts were stably transfected with a reporter gene (firefly luciferase) for bioluminescence imaging (BLI). First, we examined the role of oxidative stress in cells under hypoxic conditions. Subsequently, stem cells were transplanted to the myocardium of rats using high-resolution ultrasound, and their survival was monitored daily using BLI.Under hypoxia, oxidative stress was increased together with decreased cell survival compared to control cells, both of which were preserved by antioxidants. In living subjects, oxidative stress blockade increased early cell survival after transplantation to the myocardium, compared to untreated cells/animals.Modulation of the local microenvironment (with antioxidants) improves stem cell survival. Increased understanding of the interaction between stem cells and their microenvironment will be critical to advance the field of regenerative medicine.
View details for DOI 10.1007/s11307-009-0274-4
View details for Web of Science ID 000277375300011
View details for PubMedID 20013064
Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals
2009; 252 (1): 117-127
To determine if magnetotactic bacteria can target tumors in mice and provide positive contrast for visualization using magnetic resonance imaging.The ability of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1 (referred to from here as AMB-1), to confer positive magnetic resonance imaging contrast was determined in vitro and in vivo. For the latter studies, AMB-1 were injected either i.t. or i.v. Bacterial growth conditions were manipulated to produce small (approximately 25-nm diameter) magnetite particles, which were observed using transmission electron microscopy. Tumor targeting was confirmed using 64Cu-labeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumor.We show that AMB-1 bacteria with small magnetite particles generate T1-weighted positive contrast, enhancing in vivo visualization by magnetic resonance imaging. Following i.v. injection of 64Cu-labeled AMB-1, positron emission tomography imaging revealed increasing colonization of tumors and decreasing infection of organs after 4 hours. Viable cell counts showed that, by day 6, the bacteria had colonized tumors but were cleared completely from other organs. Magnetic resonance imaging showed a 1.22-fold (P = 0.003) increased positive contrast in tumors on day 2 and a 1.39-fold increase (P = 0.0007) on day 6.Magnetotactic bacteria can produce positive magnetic resonance imaging contrast and colonize mouse tumor xenografts, providing a potential tool for improved magnetic resonance imaging visualization in preclinical and translational studies to track cancer.
View details for DOI 10.1158/1078-0432.CCR-08-3206
View details for Web of Science ID 000269024900019
View details for PubMedID 19671860
Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation
MOLECULAR IMAGING AND BIOLOGY
2009; 11 (3): 178-187
To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs.Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.
View details for DOI 10.1148/radiol.2513081616
View details for Web of Science ID 000268362900015
View details for PubMedID 19366903
In Vivo Serial Evaluation of Superparamagnetic Iron-Oxide Labeled Stem Cells by Off-Resonance Positive Contrast
MAGNETIC RESONANCE IN MEDICINE
2008; 60 (6): 1269-1275
In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days).Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.
View details for DOI 10.1007/s11307-008-0182-z
View details for Web of Science ID 000265686900005
View details for PubMedID 19034584
Imaging of VEGF receptor in a rat myocardial infarction model using PET
JOURNAL OF NUCLEAR MEDICINE
2008; 49 (4): 667-673
MRI is emerging as a diagnostic modality to track iron-oxide-labeled stem cells. This study investigates whether an off-resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc-mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron-oxide labeled luc-mESC (SPIO-luc-mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal- and contrast-to-noise of the SPIO-luc-mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO-luc-mESC and OR signal was observed immediately after transplantation (R(2) = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO-labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability.
View details for DOI 10.1002/mrm.21816
View details for Web of Science ID 000261225100001
View details for PubMedID 19030159
Monitoring of the biological response to murine Hindlimb ischemia with Cu-64-labeled vascular endothelial growth factor-121 positron emission tomography
2008; 117 (7): 915-922
Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject.In Sprague-Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3).Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 +/- 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 +/- 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels.We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.
View details for DOI 10.2967/jnumed.107.040576
View details for Web of Science ID 000254813600028
View details for PubMedID 18375924
Reporter gene imaging following percutaneous delivery in swine - Moving toward clinical applications
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
2008; 51 (5): 595-597
US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice
2008; 246 (2): 508-518
Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training.64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61+/-0.17% ID/g (percentage of injected dose per gram) to 1.62+/-0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59+/-0.14% ID/g at day 29), and correlated with gamma-counting (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice.64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.
View details for DOI 10.1161/CIRCULATIONAHA.107.733220
View details for Web of Science ID 000253428100008
View details for PubMedID 18250264
In vivo optical bioluminescence imaging of collagen-supported cardiac cell grafts
JOURNAL OF HEART AND LUNG TRANSPLANTATION
2007; 26 (3): 273-280
To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance.In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types.US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.
View details for DOI 10.1148/radio1.2462070536
View details for Web of Science ID 000252796300021
View details for PubMedID 18180339
Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts
2006; 114: I174-I180
Histology-based survival assessment of cell grafts does not allow for in vivo follow-up. In this study we introduce two new experimental models for longitudinal in vivo survival studies of cardiac cell grafts using optical bioluminescence imaging.H9c2 cardiomyoblasts expressing both firefly luciferase (fluc) and green fluorescent protein (GFP) reporter genes were implanted into Lewis rats. In Model 1, H9c2-fluc-IRES-GFP cells (0.5 x 10(6)) were implanted into a cryoinjured abdominal wall muscle. Cells were injected using either liquid collagen (Matrigel [MG]) or phosphate-buffered saline (PBS) suspension. Cell survival was evaluated in vivo using bioluminescence imaging on days 1, 5 and 10 post-operatively. In model 2, rats underwent ligation of the left anterior descending (LAD) artery. The donor hearts were harvested, and the infarcted region was restored ex situ using 1 x 10(6) H9c2-fluc-IRES-GFP cells seeded in collagen matrix (Gelfoam [GF]) or suspended in PBS (n = 8/group). Hearts were then transplanted into the abdomen of syngeneic recipients. Optical bioluminescence imaging was performed on Days 1, 5, 8 and 14 post-operatively. After 4 weeks, immunohistologic studies were performed.For model 1, at day 5, bioluminescence signals were markedly higher for the H9c2/MG group (449 +/- 129 photons/second x 10(3)) compared with the H9c2/PBS group (137 +/- 82 photons/second x 10(3)) (p < 0.05). For model 2, bioluminescence signals were significantly (p < 0.04) higher in the H9c2/GF group compared with plain cell injection on days 5 (534 +/- 115 vs 219 +/- 34) and 8 (274 +/- 34 vs 180 +/- 23). Data were in accordance with GFP immunohistology.Optical bioluminescence is a powerful method for assessment of cardiac cell graft survival in vivo. Collagen matrices support early survival of cardiomyoblasts after transplantation into injured musculature.
View details for Web of Science ID 000244979000010
View details for PubMedID 17346630
Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts
2006; 114: I167-I173
Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts.H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1x10(6) mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a "working heart" model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21+/-0.03; mH9c2: 0.21+/-0.04; control: 0.15+/-0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0+/-6.2; mH9c2: 48.7+/-6.1; control: 34.3+/-6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology.Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.
View details for DOI 10.1161/CIRCULATIONAHA.105.001370
View details for Web of Science ID 000238688200029
View details for PubMedID 16820569
Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival
MOLECULAR IMAGING AND BIOLOGY
2006; 8 (3): 163-170
Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices.H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1 x 10(6) genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 microg/mL), or (5) GF/MG/FGF (10 microg/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function.Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.
View details for DOI 10.1161/CIRCULATIONAHA.105.001297
View details for Web of Science ID 000238688200028
View details for PubMedID 16820568
Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation
2006; 25 (1): 29-38
The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death. PROCEDURES AND RESULTS: Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3x10(6)) were injected into thigh muscles of Sprague-Dawley rats (N=30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day -3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0+/-12.1% (dexamethasone), 30.5+/-12.5% (tacrolimus), and 21.5+/-3.5% (mycophenolate) vs. 12.0+/-5.0% (control) and 8.3+/-5.0% (cyclosporine) at day 4 (P<0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.
View details for DOI 10.1007/s11307-006-0038-3
View details for Web of Science ID 000237754300003
View details for PubMedID 16555032
Image-guided cardiac cell delivery using high-resolution small-animal ultrasound
2005; 12 (6): 1142-1147
Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.
View details for DOI 10.1152/physiolgenomics.00254.2005
View details for Web of Science ID 000236722700004
View details for PubMedID 16390873
Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium
2004; 110 (6): 685-691
Open-chest cardiac injection is the preferred delivery method for cardiac gene and stem cell therapy in small animals, but it is invasive and the operator is unable to see the actual delivery site. High-resolution ultrasound has recently been developed for small-animal imaging. We tested the hypothesis that image-guided cardiac cell delivery using high-resolution ultrasound guidance is feasible and reproducible. Sprague-Dawley rats (n = 11) were imaged using high-resolution ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After injection, bioluminescence imaging was performed using a cooled charged-coupled camera. Injection was successful in all animals and was associated with no mortality. The signal detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03) and highly correlated with ex vivo assays (R(2) = 0.82). In addition, the optical signal could be followed longitudinally using bioluminescence imaging. Ultrasound image-guided cardiac cell delivery is an effective, safe, and reproducible way to perform cell delivery to a specific myocardial region and can be combined with assessment of cardiac function. We are confident that the use of these technologies will play a significant role in the future of gene and cell therapy.
View details for DOI 10.1016/j.ymthe.2005.07.532
View details for Web of Science ID 000233864700017
View details for PubMedID 16111921
Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery
2004; 109 (11): 1415-1420
Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro-positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system.Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1x10(10) pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging.Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.
View details for DOI 10.1161/01.CIR.0000138153.02213.22
View details for Web of Science ID 000223194700008
View details for PubMedID 15302807
Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography
2003; 108 (11): 1302-1305
We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES).Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart.The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.
View details for DOI 10.1161/01.CIR.0000121727.59564.5B
View details for Web of Science ID 000220364700015
View details for PubMedID 15007006
Regulatory proteins alter nucleotide binding to acto-myosin of sliding filaments in motility assays
2003; 85 (2): 1046-1052
The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques.For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3x10(6) to 5x10(5)) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3x10(6) cells: day 1 (627 000+/-15%), day 2 (346 100+/-21%), day 4 (112 800+/-20%), day 8 (78 860+/-24%), day 12 (67 780+/-12%), and day 16 (62 200+/-5% p x s(-1) x cm(2-1) x sr(-1)). For micro-positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3x10(6)) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48+/-0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01+/-0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays.The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.
View details for DOI 10.1161/01.CIR.0000091252.20010.6E
View details for Web of Science ID 000185328800006
View details for PubMedID 12963637
The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.
View details for Web of Science ID 000184428300033
View details for PubMedID 12885651